New-generation pathology-testing devices are examples of microtechnological systems where small quantities (nanoliters) of liquid need to be mixed. Presently, much larger volumes (milliliters) of a patient's body fluids are sent away for testing in pathology laboratories, where such samples are mixed with reagents indicating particular medical conditions. This causes significant delays between test and diagnosis. Thus there is great interest in cheap, self-contained ‘lab-on-a-chip’ micro-devices that could make instant screening tests at the point of care. Because of their size, such devices inherently demand the mixing of only a tiny drop. Unfortunately, mixing at microscale is very difficult and slow, owing to the absence of turbulence.
The absence of turbulence at microscale makes most proposed point-of-care or handheld pathology screening tests dependent on diffusion, which takes an impractically long time before a result can be obtained. In this application, very small, finite volumes of liquid, typically a few tens of microliters, must be set into motion. A small drop two millimeters in diameter has a volume of about four microliters. The starting volumes are small and finite for two reasons. Firstly, obtaining milliliters rather than microliters from a patient escalates the intervention required—instead of a pinprick or swab, a syringe puncturing a vein would be required, demanding paramedical assistance and impractically lengthening any mass-screening or point-of-care procedure. Secondly, at least the part of the device in contact with the sample should be disposable to eliminate expensive sterilization and cross-contamination issues, and micro-volumes allow cheap, disposable sample-processing elements. In some cases, one or more microliter-sized drops must be mixed. In other cases, detection of a target molecule relies on target molecules in the liquid being driven on a sufficiently space-filling trajectory for it to contact to the detector surface in as short a time as possible. In such ‘batch’ processes, most typical of pathology tests, engineering of complex devices to pump or stir the liquids together could make the device uneconomic.
An application typical of the new generation of screening-type pathology tests is to mix a single drop of patient's sample fluid with a pre-loaded ‘reagent’ or detector liquid, which would most probably contain engineered antibodies used in an immunoassay. The sample could be taken from a pinprick or swab, and might be collected in a context such as an airport, or a patient's point-of-care, where an elementary ‘maybe/no’ screening result is needed in a short time. The mixing should occur on a ‘chip’ that is simple and cheap enough to be disposable, so that any complex electronics are housed in a separate, ‘reader’ unit. If the target protein indicating a disease state is present in the patient's sample, the antibodies would bind to it and undergo a colour or fluorescence change, or cause an optical change in the properties of the surface to which antibodies may be bound. This change would be detected by equipment in the reader unit. The challenge is to design a chip that is capable of receiving a single drop, without a complex liquid feeding system, and mix the drop efficiently enough with the reagent for any binding reactions to occur over a timescale of tens of seconds rather than hours. Timescales of hours would render the test useless from a point-of-care perspective.
There are many aspects to mixing in microfluidic systems, as reviewed recently by Ottino & Wiggins1, and there are many relevant quantitative measures of mixing2. From a practical perspective, whatever method is used to achieve micromixing3-6 is likely to be subjected to a series of engineering refinements and improvements, especially in the light of the final end-use of the device. Presently, most micromixing efficiency assessments rely on simple assessments by eye of the mixing time; a recent example of batch micromixing which makes such an assessment is Lee & Ram7. In general, the mixing time is the time for introduced dye to appear uniformly dispersed through a mixing vessel8,9.
Acoustic microstreaming is a phenomenon where sound waves propagating around a small object create a mean flow in the vicinity of the obstacle. It is a nonlinear second-order effect driven by the viscous shear in the boundary layer near the object. It is particularly enhanced where the object is a bubble, because the bubble can resonate to the applied sound in one or more ways, locally amplifying and transforming the microstreaming effect. Bubbles can oscillate in several ways, the most common being a volumetric or ‘breathing’ mode10, which has a well-known natural frequency inversely proportional to the bubble radius. However, bubbles in microdevices are invariably near at least one wall, which is known to make a significant change to the volumetric mode frequency11. Moreover, under the influence of applied sound, the bubble can also translate either parallel to or perpendicular to walls, complicating the flow12. Thus, although experiments on streaming around an oscillating bubble date to the 1950s13,14, rigorous theoretical prediction of the flows in practical geometries remains challenging. More recently, Marmottant and Hilgenfeldt15 showed that the shear forces developed around a microstreaming bubble could be used to lyse cells.
Liu et al.3,16 showed that acoustic microstreaming could be used for micromixing. In their experiment, air bubbles trapped in pockets inside a circular chamber 300 μm deep and 15 mm in diameter were excited; dye was used to observe the resulting streaming motion. However, there is little quantitative information available about the flow field induced by the streaming and mixing times were subjectively estimated.
US 2003/0175947 discloses a device which utilises one or more bubbles caused to oscillate at a resonant frequency in order to facilitate mixing by a process of cavitation microstreaming. The principle theory presented is that the microstreaming occurs when the relevant bubble undergoes volume change within a sound field. Although there is a disclosure of the applied frequency being over a frequency range, the teaching of the reference is that microstreaming, arising about a single bubble excited close to resonance, produces strong liquid circulation flow in the associated microfluidic chamber but that a variation in frequency or radius of the bubble from the conditions for maximum motion causes the streaming to be inappreciable.